Accumulating evidence suggest that many human cancers consist of functionally heterogeneous cells organized in a hierarchical manner with less mature cancer stem cells (CSC) giving rise to differentiated tumor cells. The distinction between these cell types has important implications for the development of effective anti- cancer strategies as mature tumor cells that form the majority of cells and phenotypically characterize the disease lack long-term replicative potential. In contrast, rare CSC appear to arise from the malignant transformation of normal stem cells or progenitors and retain the capacity to both self-renew and produce mature progeny. Therefore, CSC are thought to possess the growth potential to reform tumors that is clinically recognized as disease relapse or progression. We have studied CSC in multiple myeloma (MM), a disease characterized by the clonal expansion of neoplastic plasma cells. Although plasma cells are the hallmark of the disease, we found that these cells are terminally differentiated and have limited replicative potential. Instead, MM plasma cells arise from a self-renewing CSC compartment that phenotypically resembles normal memory B cells. Our preliminary data demonstrate that MM CSC are relatively resistant to drugs currently used to clinically treat the disease and biologically distinct from MM plasma cells;these data may explain the typical clinical response pattern of MM patients to treatment (i.e., initial response followed by relapse and disease progression). Furthermore, we have found that cellular pathways which regulate cell fate decisions in normal stem cells are similarly active in MM CSC. We hypothesize that CSC are responsible for the long-term outcomes of patients with MM and that effective stem cell targeted strategies will lead to long-term remissions. Accordingly, we propose to: 1). Further characterize MM CSC and study their clinical relevance;and 2). Study the role of developmental signaling pathways in the regulation of MM CSC and their potential as therapeutic targets. Project Narrative We previously demonstrated that clonogenic cells in multiple myeloma are not plasma cells that are typically associated with the disease, but rather mature B cells that appear to have the capacity to undergo self-renewal and give rise to terminally differentiated tumor cells (i.e., plasma cells). We propose to further characterize myeloma stem cells by correlating their frequency and growth potential with validated prognostic factors that predict clinical outcomes as well as investigate the role of developmental signaling pathways that include Hedgehog, Notch and Wnt, in regulating myeloma cancer stem cells.